Fc fusion proteins of human granulocyte colony-stimulating factor with increased biological activities

ABSTRACT

Fc fusion proteins of human G-CSF with increased biological activities relative to rhG-CSF on a molar basis are disclosed. The hG-CSF-L-vFc fusion protein comprises hG-CSF, a flexible peptide linker of about 20 or fewer amino acids, and a human IgG Fc variant. The Fc variant is of a non-lytic nature and shows minimal undesirable Fc-mediated side effects. A method is also disclosed to make or produce such fusion proteins at high expression levels. Such hG-CSF-L-vFc fusion proteins exhibit extended serum half-life and increased biological activities, leading to improved pharmacokinetics and pharmacodynamics, thus fewer injections will be needed within a period of time.

BACKGROUND

[0001] Granulocyte colony-stimulating factor (G-CSF) is a 20 kilodalton(kDa) glycoprotein that promotes the proliferation of progenitor cellsand induces their differentiation into neutrophils. In addition, G-CSFprolongs the survival of mature neutrophils and activates theirfunctions Human G-CSF (hG-CSF) is produced by monocytes, macrophages,fibroblasts and endothelial cells (see, for example, Moore, Annu. Rev.Immunol, 9:159-191, 1991; Nicola, Annu. Rev. Biochem., 58:45-77, 1991).The biological effects of G-CSF are mediated through its interactionwith the G-CSF receptor (G-CSF-Rc) expressed on the surface of bonemarrow hematopoietic progenitors and cells of the myeloid lineage. Uponbinding G-CSF, the receptor is activated and undergoes homodimerization,followed by phosphorylation of Janus family of tyrosine kinases.Subsequently, a series of intracellular signal transduction events takeplace, leading to the increase of the number of progenitor cells, theirmaturation into neutrophils, and further activation of effectorfunctions in mature neutrophils (see, for example, Demetri et al.,Blood, 78:2791-2808, 1991). Therefore, G-CSF plays an essential role notonly in the regulation and maintenance of hematopoiesis, but also inhost defense against infection and inflammation.

[0002] Recombinant human G-CSF (rhG-CSF) is widely used in the treatmentof patients with neutropenia as a result of receiving chemotherapy.Administration of rhG-CSF is effective in restoring functioningneutrophils to these patients, leading to a decrease ofinfection-related events. Use of rhG-CSF allows intensified dosing orscheduling of chemotherapeutic agents that may be of benefit to cancerpatients. Besides chemotherapy-induced neutropenia, rhG-CSF has beenused for the treatment of myelosuppression after bone marrowtransplantation, acute leukemia, aplastic anemia, myelodysplasticsyndrome, severe chronic neutropenias, and mobilization of peripheralblood progenitor cells for transplantation (see, for example, Welte etal., Blood, 88:1907-1929, 1996).

[0003] The elimination half-life of the serum concentration of rhG-CSFis approximately 3 to 4 h for intravenous or subcutaneousadministration. The safety profile and patient tolerance of rhG-CSF aregood with medullary bone pain being the only frequent and significantside effect. The relatively low toxicity of rhG-CSF has made it feasibleto develop longer-acting derivatives to decrease the inconvenience ofthe daily or twice-daily injection schedule. Attachment of polyethyleneglycol (PEG) to various proteins, including G-CSF, has been reported toyield derivatives with higher in vivo potency due to their longerhalf-lives (see, for example, Zalipsky et al., in “PEG chemistry:biotechnical and biomedical applications”, pp. 347-370, 1992).PEG-conjugated proteins usually have considerably lower in vitrobiological activity than their unmodified parent proteins (Eliason etal., Stem Cells, 18:40-45, 2000). The increased in vivo potency of thesemodified proteins is, at least in part, due to decreased removal by thekidney in a manner proportional to their molecular weight (Yamaoda etal., J. Pharmaceut. Sci., 83:601-606, 1994). We unexpectedly discoverthat it is possible to increase the potency of hG-CSF through prolongingits half-life as well as enhancing its biological activity is to attachthe Fc region derived from human IgG at the C-terminus of hG-CSF, asdescribed in this invention.

[0004] Immunoglobulins of IgG class are among the most abundant proteinsin human blood. Their circulation half-lives can reach as long as 21days. Fusion proteins have been reported to combine the Fc regions ofIgG with the domains of another protein, such as various cytokines andsoluble receptors (see, for example, Capon et al., Nature, 337:525-531,1989; Chamow et al., Trends Biotechnol., 14:52-60, 1996); U.S. Pat. Nos.5,116,964 and 5,541,087). The prototype fusion protein is a homodimericprotein linked through cysteine residues in the hinge region of IgG Fc,resulting in a molecule similar to an IgG molecule without the CHIdomains and light chains. Due to the structural homology, Fc fusionproteins exhibit in vivo pharmacokinetic profile comparable to that ofhuman IgG with a similar isotype. This approach has been applied toseveral therapeutically important cytokines, such as IL-2 andIFN-α_(2a), and soluble receptors, such as TNF-Rc and IL-5-Rc (see, forexample, U.S. Pat. Nos. 5,349,053 and 6,224,867). It is desirable toextend the circulating half-life of G-CSF and/or to increase itsbiological activity by making fusion proteins containing G-CSF linked tothe Fc portion of the human IgG protein as disclosed and/or described inthis invention.

[0005] Erythropoietin (EPO) derivatives, such as dimers, have beenreported. Relative to the EPO monomer, a fusion protein consisting oftwo complete EPO domains separated by a 3- to 7-amino acid peptidelinker exhibited reduced activity (Qiu et al., J. Biol. Chem.,273:11173-11176, 1998). However, when the peptide linker between the twoEPO domains was 17 amino acids in length, the dimeric EPO moleculeexhibited considerably enhanced in vitro and in vivo activities (see,for example, Sytkowski et al., J. Biol. Chem., 274:24773-24778, 1999;U.S. Pat. No. 6,187,564 ). The length of the peptide linker between thetwo hematopoietic growth factors is important, while not bound by thistheory, presumably due to its effect on the flexibility of suchmolecular forms. We find that this approach is generally applicable toother therapeutic proteins, including G-CSF. We'll also refer this tothis as a flexible peptide linker.

[0006] In most of the reported Fc fusion protein molecules, a hingeregion serves as a spacer between the Fc region and the cytokine orsoluble receptor at the amino-terminus, allowing these two parts of themolecule to function separately (see, for example, Ashkenazi et al.,Current Opinion in Immunology, 9:195-200, 1997). A human G-CSF fusionprotein with an appropriate peptide linker between the hG-CSF and Fcmoieties (hG-CSF-L-Fc) is more active than rhG-CSF, with in vitroactivity at least 2-fold as that of rhG-CSF on a molar basis. It isdiscovered according to this invention that an added peptide linkerpresent between hG-CSF and a human IgG Fc variant enhances the in vitrobiological activity of the hG-CSF-L-Fc molecule in two ways: (1) keepingthe Fc region away from the G-CSF-Rc binding sites on G-CSF, and (2)keeping one G-CSF from the other G-CSF domain, so both G-CSF domains caninteract with G-CSF-Rc on the granulocyte precursor cells independently.For the present invention, a flexible peptide linker of about 20 orfewer amino acids in length is preferred. More preferably, the peptidelinker should have at least two amino acids in length. Furthermore, itis even more preferable to use a peptide linker comprising two or moreof the following amino acids: glycine, serine, alanine, and threonine.

[0007] The Fc region of human immunoglobulins plays a significant rolein immune defense for the elimination of pathogens. Effector functionsof IgG are mediated by the Fc region through two major mechanisms: (1)binding to the cell surface Fc receptors (Fc_(γ)Rs) can lead toingestion of pathogens by phagocytosis or lysis by killer cells via theantibody-dependent cellular cytotoxicity (ADCC) pathway, or (2) bindingto the C1q part of the first complement component C1 initiates thecomplement-dependent cytotoxicity (CDC) pathway, resulting in the lysisof pathogens. Among the four human IgG isotypes, IgG1 and IgG3 areeffective in binding to Fc_(γ)R. The binding affinity of IgG4 to Fc_(γ)Ris an order of magnitude lower than that of IgG1 or IgG3, while bindingof IgG2 to Fc_(γ)R is below detection. Human IgG1 and IgG3 are alsoeffective in binding to C1q and activating the complement cascade. HumanIgG2 fixes complement poorly, and IgG4 appears quite deficient in theability to activate the complement cascade (see, for example, Jefferiset al., Immunol. Rev., 163:59-76, 1998). For therapeutic use in humans,it is essential that when hG-CSF-L-Fc binds to G-CSF-Rc on the surfaceof the progenitor cells or other cells of the myeloid lineage, the Fcregion of the fusion protein will not mediate undesirable effectorfunctions, leading to the lysis or removal of these cells. Accordingly,the Fc region of hG-CSF-L-Fc must be of a non-lytic nature, i.e. the Fcregion must be inert in terms of binding to Fc_(γ)Rs and C1q for thetriggering of effector functions. It is clear that none of the naturallyoccurring IgG isotypes is suitable for use to produce the hG-CSF-L-Fcfusion protein. To obtain a non-lytic Fc, certain amino acids of thenatural Fc region have to be mutated for the attenuation of the effectorfunctions.

[0008] By comparing amino acid sequences of human and murine IgGisotypes, a portion of Fc near the N-terminal end of the CH2 domain isimplicated to play a role in the binding of IgG Fc to Fc_(γ)Rs. Theimportance of a motif at positions 234 to 237 has been demonstratedusing genetically engineered antibodies (see, for example, Duncan etal., Nature, 332:563-564, 1988). The numbering of the amino acidresidues is according to the EU index as described in Kabat et al. (inSequences of Proteins of Immunological Interest, 5^(th) Edition, UnitedStates Department of Health and Human Services, 1991). Among the fourhuman IgG isotypes, IgG1 and IgG3 bind Fc_(γ)Rs the best and share thesequence Leu234-Leu-Gly-Gly237 (only IgG1 is shown in FIG. 1). In IgG4,which binds Fc_(γ)Rs with a lower affinity, this sequence contains asingle amino acid substitution, Phe for Leu at position 234. In IgG2,which does not bind Fc_(γ)Rs, there are two substitutions and a deletionleading to Val234-Ala-Gly237 (FIG. 1). To minimize the binding of Fc toFc_(γ)R and hence the ADCC activity, Leu235 in IgG4 has been replaced byAla (see, for example, Hutchins et al., Proc. Natl. Acad. Sci. USA,92:11980-11984, 1995). IgG1 has been altered in this motif by replacingGlu233-Leu-Leu235 with Pro233-Val-Ala235, which is the sequence fromIgG2. This substitution resulted in an IgG1 variant devoid ofFc_(γ)R-mediated ability to deplete target cells in mice (see, forexample, Isaacs et al., J. Immunol., 161:3862-3869, 1998).

[0009] A second portion that appears to be important for both Fc_(γ)Rand C1q binding is located near the carboxyl-terminal end of CH2 domainof human IgG (see, for example, Duncan et al., Nature, 332:738-740,1988). Among the four human IgG isotypes, there is only one site withinthis portion that shows substitutions: Ser330 and Ser331 in IgG4replacing Ala330 and Pro331 present in IgG1, IgG2, and IgG3 (FIG. 1).The presence of Ser330 does not affect the binding to Fc_(γ)R or C1q.The replacement of Pro331 in IgG1 by Ser virtually abolished IgG1ability to C1q binding, while the replacement of Ser331 by Pro partiallyrestored the complement fixation activity of IgG4 (see, for example, Taoet al., J. Exp. Med., 178:661-667, 1993; Xu et al., J. Biol. Chem.,269:3469-3474, 1994).

[0010] We discover that at least three Fc variants (vFc) can be designedand/or used for the production of hG-CSF-L-vFc fusion proteins (FIG. 1).Human IgG2 Fc does not bind Fc_(γ)R but showed weak complement activity.An Fc_(γ2) variant with Pro331 Ser mutation should have less complementactivity than natural Fc_(γ2) while remain as a non-binder to Fc_(γ)R.IgG4 Fc is deficient in activating the complement cascade, and itsbinding affinity to Fc_(γ)R is about an order of magnitude lower thanthat of the most active isotype, IgG1. An Fc_(γ4) variant with Leu235Alamutation should exhibit minimal effector functions as compared to thenatural Fc_(γ4). The Fc_(γ1) variant with Leu234Val, Leu235Ala andPro331Ser mutations also will exhibit much less effector functions thanthe natural Fc_(γ1). These Fc variants are more suitable for thepreparation of the G-CSF fusion proteins than naturally occurring humanIgG Fc. It is possible that other replacements can be introduced for thepreparation of a non-lytic Fc without compromising the circulatinghalf-life or causing any undesirable conformational changes.

[0011] There are many advantages with the present invention. Theincreased activity and prolonged presence of the hG-CSF-L-vFc fusionprotein in the serum can lead to lower dosages as well as less frequentinjections. Less fluctuations of the drug in serum concentrations alsomeans improved safety and tolerability. Less frequent injections mayresult in better patient compliance and quality of life. ThehG-CSF-L-vFc fusion protein containing a non-lytic Fc variant willtherefore contribute significantly to the management of a variety ofconditions associated with an impaired immune or hematopoietic system,including cancer chemotherapy, leukemias, anemias AIDS, bone marrowtransplantation, and chronic neutropenias.

SUMMARY OF THE INVENTION

[0012] One aspect of the present invention relates to an hG-CSF-L-vFcfusion protein. This hG-CSF-L-vFc fusion protein comprises hG-CSF, apeptide linker (denoted by L), and a human IgG Fc variant (denoted byvFc). It is preferable to use a flexible peptide linker of about 20 orfewer, more preferably to about 2, amino acids in length and theflexible peptide linker contains or comprises of two or more of aminoacids selected from the group consisting of glycine, serine, alanine,and threonine. The IgG Fc variant is of non-lytic nature and containsamino acid mutations as compared to naturally occurring IgG Fc.

[0013] It is another embodiment of the present invention that the humanIg Fc comprises a hinge, CH2, and CH3 domains of human IgG, such ashuman IgG1, IgG2, and IgG4. The CH2 domain contains amino acid mutationsat positions 228, 234, 235, and 331 (defined by the EU numberingsystem). It is believed that these amino acid mutations serve toattenuate the effector functions of Fc.

[0014] In yet another embodiment of the present invention, a method isdisclosed for making or producing such recombinant fusion proteins froma mammalian cell line such as a CHO-derived cell line. Growingtransfected cell lines under conditions such that the recombinant fusionprotein is expressed in its growth medium in excess of 10, preferably30, μg per million cells in a 24 hour period. These hG-CSF-L-vFc fusionproteins are characterized by and exhibit increased/enhanced biologicalactivity, preferably at least two fold (2×) in vitro activity, on amolar basis, relative to that of rhG-CSF and extended serum half-lifewithout undesirable side effects, leading to improved pharmacokineticsand pharmacodynamics, thus lower dosages and fewer injections would beneeded to achieve similar efficacies.

[0015] A further embodiment of the present invention provides a methodfor making a recombinant fusion protein comprising hG-CSF, a flexiblepeptide linker, and a human IgG Fc variant, which method comprises: (a)generating a CHO-derived cell line; (b) growing the cell line underconditions the recombinant fusion protein is expressed in its growthmedium in excess of 10 μg, preferably 30 μg, per million (10⁶) cells ina 24 hour period; and (c) purifying the expressed protein from step (b),wherein the recombinant fusion protein is characterized by and exhibitsan enhanced in vitro biological activity of at least 2 fold (2×)relative to that of rhG-CSF on a molar basis. In this case, preferably,the flexible peptide linker containing or comprising about 20 or fewer,but not fewer than 2, amino acids is present between hG-CSF and thehuman IgG Fc variant; and the flexible peptide linker comprises two ormore amino acids selected from the group consisting of glycine, serine,alanine, and threonine; and wherein the human IgG Fc variant comprises ahinge, CH2, and CH3 domains selected from the group consisting of humanIgG2 with Pro331Ser mutation, human IgG4 with Ser228Pro and Leu235Alamutations, and human IgG1 with Leu234Val, Leu235Ala, and Pro331Sermutations.

BRIEF DESCRIPTIONS OF THE DRAWINGS

[0016]FIG. 1 shows the amino acid sequence alignment from the hinge andCH2 regions of human IgG1, IgG2, IgG4 and their variants. Three portionsare compared: amino acid position 228, 234-237, and 330-331. Amino acidmutations of the variants are indicated in bold italics. The EUnumbering system is used for the amino acid residues.

[0017]FIG. 2 shows the nucleotide sequence and deduced amino acidsequence of (A) hG-CSF-L-vFc_(γ2), (B) hG-CSF-L-vFc_(γ4), and (C)hG-CSF-L-vFc_(γ1) as the HindIII-EcoRI fragment in the respective phGFPexpression vector. Amino acid residues −30 to −1 is the leader peptideof human G-CSF. The mature protein contains human G-CSF (amino acidresidues 1 to 174), a peptide linker (amino acid residues 175 to 190),and a Fc variant (amino acid residues 191 to 418 of vFc_(γ2), 191 to 419of vFc_(γ4), and 191 to 417 of vFc_(γ1)). In the Fc regions, nucleotideand corresponding amino acid mutations in bold are also underlined.

DETAILED DESCRIPTION OF THE INVENTION

[0018] 1. Construction of the Gene Encoding the hG-CSF-L-vFc_(γ2) FusionProtein

[0019] A fusion protein is assembled from several DNA segments. The geneencoding the leader peptide and mature protein of human G-CSF isobtained by reverse transcription and polymerase chain reaction (PCR)using RNA prepared from the human bladder carcinoma 5637 cell line. Forthe convenience of cloning, SEQ ID NO:1 (Table 1), which incorporates arestriction enzyme cleavage site (HindIII) is used as the 5′oligonucleotide primer. Table 1 shows the sequences of oligonucleotidesused for the cloning of the hG-CSF-L-vFc fusion proteins. The 3′ primer(SEQ ID NO:2) eliminates the G-CSF termination codon and incorporates aBamHI site. The resulting DNA fragments of approximately 600 bp inlength are inserted into a holding vector such as pUC19 at the HindIIIand BamHI sites to give the phGCSF plasmid. The sequence of the humanG-CSF gene is confirmed by DNA sequencing.

[0020] The gene encoding the Fc region of human IgG2 (Fc_(γ2)) isobtained by reverse transcription and PCR using RNA prepared from humanleukocytes and appropriate 5′ (SEQ ID NO:3) and 3′ (SEQ ID NO:4)primers. Resulting DNA fragments of Fc_(γ2) containing completesequences of the hinge, CH2, and CH3 domains of IgG2 will be used as thetemplate to generate the Fc_(γ2) Pro331Ser variant (vFc_(γ2)) in whichPro at position 331 of Fc_(γ2) is replaced with Ser. To incorporate thismutation, two segments are produced and then assembled by using thenatural Fc_(γ2) as the template in overlapping PCR. The 5′ segment isgenerated by using SEQ ID NO:3 as the 5′ primer and SEQ ID NO:5 as the3′ primer. The 3′ segment is generated by using SEQ ID NO:6 as the 5′primer and SEQ ID NO:4 as the 3′ primer. These two segments are thenjoined at the region covering the Pro331Ser mutation by using SEQ IDNO:7 as the 5′ primer and SEQ ID NO:4 as the 3′ primer. The SEQ ID NO:7primer contains sequences encoding a 16-amino acid Gly-Ser peptidelinker including a BamHI restriction enzyme site. The resulting DNAfragments of approximately 700 bp in length are inserted into a holdingvector such as pUC19 at the BamHI and EcoRI sites to give the pL-vFcγ2plasmid. The sequence of the gene is confirmed by DNA sequencing.

[0021] To prepare the hG-CSF-L-vFc_(γ2) fusion gene, the hG-CSF fragmentis excised from the phGCSF plasmid with HindIII and BamHI and ispurified by agarose gel electrophoresis. The purified fragment is theninserted to the 5′-end of the peptide linker in the pL-vFcγ2 plasmid togive the phG-CSF-L-vFcγ2 plasmid. The fusion gene comprises hG-CSF, aGly-Ser peptide linker and the Fc_(γ2) variant gene.

[0022] The presence of a peptide linker, preferably a flexible linker,between (and chemically bound to both) the hG-CSF and Fc moietiesincreases the flexibility of the hG-CSF domains and enhances itsbiological activity. For the present invention, a peptide linker ofabout 20 or fewer amino acids in length is preferred. While a singleamino acid is within the scope of the present invention, it is preferredto have a flexible peptide linker of about 20 to about 2 amino acids inlength. Peptide linker containing or comprising of two or more of aminoacids selected from the group consisting of glycine, serine, alanine,and threonine can be used preferably. An example of the peptide linkercontains Gly-Ser peptide building blocks, such as GlyGlyGlyGlySer. FIG.2A shows a fusion gene containing sequences encoding hG-CSF, a 16-aminoacid peptide linker (GlySerGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySer),and the Fc_(γ2) Pro331Ser variant.

[0023] The complete gene encoding the hG-CSF-L-vFc fusion protein isthen inserted at the HindIII and EcoRI sites of a mammalian expressionvector, such as pcDNA3 (Invitrogen). The final expression vectorplasmid, named phGFP2, contains the cytomegalovirus early genepromoter-enhancer that is required for high level expression inmammalian cells. The plasmid also contains selectable markers to conferampicillin resistance in bacteria, and G418 resistance in mammaliancells. In addition, the phGFP2 expression vector contains thedihydrofolate reductase (DHFR) gene to enable the co-amplification ofthe hG-CSF-L-vFcγ2 fusion gene and the DHFR gene in the presence ofmethotrexate (MTX) when the host cells are deficient in the DHFR geneexpression (see, for example, U.S. Pat. No. 4,399,216).

[0024] 2. Construction of the Gene Encoding the hG-CSF-L-vFc_(γ4) FusionProtein

[0025] Human IgG4 is observed partly as half antibody molecules due tothe dissociation of the inter-heavy chain disulfide bonds in the hingedomain. This is not seen in the other three human IgG isotypes. A singleamino acid substitution replacing Ser228 with Pro, which is the residuefound at this position in IgG1 and IgG2, leads to the formation of IgG4complete antibody molecules (see, for example, Angal et al., Molec.Immunol., 30:105-108, 1993; Owens et al., Immunotechnology, 3:107-116,1997; U.S. Pat. No. 6,204,007). The Fc_(γ4) variant containing Leu235Alamutation for the minimization of FcR binding will also give rise to ahomogeneous fusion protein preparation with this additional Ser228Promutation.

[0026] The gene encoding the Fc region of human IgG4 (Fc_(γ4)) isobtained by reverse transcription and PCR using RNA prepared from humanleukocytes and appropriate 5′ primer (SEQ ID NO:8) and 3′ primer (SEQ IDNO:9). Resulting DNA fragments of Fc_(γ4) containing complete sequencesof the hinge, CH2, and CH3 domains of IgG4 is used as the template togenerate the Fc_(γ4) variant with Ser228Pro and Leu235Ala mutations(vFc_(γ4)) in which Ser228 and Leu235 have been replaced with Pro andAla, respectively. The CH2 and CH3 domains are amplified using the 3′primer (SEQ ID NO:9) and a 5′ primer containing the Leu235Ala mutation(SEQ ID NO: 10). This amplified fragment, together with a syntheticoligonucleotide of 60 bases in length (SED ID NO:11) containing bothSer228Pro and Leu235Ala mutations, are joined in PCR by using SEQ IDNO:12 as the 5′ primer and SEQ ID NO:9 as the 3′ primer. The SEQ ID NO:12 primer contains sequences encoding a 16-amino acid Gly-Ser peptidelinker including the BamHI site. The resulting DNA fragments ofapproximately 700 bp in length are inserted into a holding vector suchas pUC19 at the BamHI and EcoRI sites to give the pL-vFcγ4 plasmid. Thesequence of the gene is confirmed by DNA sequencing.

[0027] To prepare the hG-CSF-L-vFc_(γ4) fusion gene, the hG-CSF fragmentis excised from the phGCSF plasmid with HindIII and BamHI and theninserted to the 5′ -end of the peptide linker in the pL-vFcγ4 plasmid togive the phG-CSF-L-vFcγ4 plasmid. This fusion gene comprising hG-CSF, a16-amino acid Gly-Ser peptide linker and the Fc_(γ4) variant gene isthen inserted at the HindIII and EcoRI sites of a mammalian expressionvector, such as pcDNA3 (Invitrogen), as described for thehG-CSF-L-vFc_(γ2) fusion protein. The final expression vector plasmid isdesignated as phGFP4. FIG. 2B shows a fusion gene containing sequencesencoding hG-CSF, a 16-amino acid peptide linker(GlySerGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySer), and the Fc_(γ4)variant with Ser228Pro and Leu235Ala mutations.

[0028] 3. Construction of the Gene Encoding the hG-CSF-L-vFc_(γ1) FusionProtein

[0029] The hinge domain of human IgG1 heavy chain contains 15 amino acidresidues (GluProLysSerCysAspLysThrHisThrCysProProCysPro) including 3cysteine residues. Out of these 3 cysteine residues, the 2^(nd) and3^(rd) are involved in the formation of disulfide bonding between twoheavy chains. The 1^(st) cysteine residue is involved in the disulfidebonding to the light chain of IgG. Since there is no light chain presentin the Fc fusion protein molecule, this cysteine residue may pair withother cysteine residues, leading to nonspecific disulfide bonding. Thehinge domain of Fc_(γ1) can be truncated to eliminate the 1^(st)cysteine residue (AspLysThrHisThrCysProProCysPro). The gene encoding theFc_(γ1) region is obtained by reverse transcription and PCR using RNAprepared from human leukocytes and appropriate 5′ primer (SEQ ID NO:13)and 3′ primer (SEQ ID NO:4). Resulting DNA fragments containing thetruncated hinge and complete sequences of CH2 and CH3 domains of Fc_(γ1)is used as the template to generate the Fc_(γ1) variant with Leu234Val,Leu235Ala, and Pro331Ser mutations (vFc_(γ1)).

[0030] One way to incorporate these mutations is as follows: twosegments are produced and then assembled by using the natural Fc_(γ1) asthe template in overlapping PCR. The 5′ segment is generated by usingSEQ ID NO:14 as the 5′ primer and SEQ ID NO:5 as the 3′ primer. This 5′primer contains the Leu234Val, Leu235Ala mutations and the 3′ primercontains the Pro331Ser mutation. The 3′ segment is generated by usingSEQ ID NO:6 as the 5′ primer and SEQ ID NO:4 as the 3′ primer. These 5′and 3′ segments are then joined at the region covering the Pro331Sermutation by using SEQ ID NO:14 as the 5′ primer and SEQ ID NO:4 as the3′ primer. This amplified fragment of approximately 650 bp in length,together with a synthetic oligonucleotide of 55 bases (SED ID NO:15)containing Leu234Val and Leu235Ala, are joined in PCR by using SEQ IDNO:16 as the 5′ primer and SEQ ID NO:4 as the 3′ primer. The SEQ IDNO:16 primer contains sequences encoding a 16-amino acid Gly-Ser peptidelinker including the BamHI site. The resulting DNA fragments ofapproximately 700 bp in length are inserted into a holding vector suchas pUC19 at the BamHI and EcoRI sites to give the pL-vFc_(γ1) plasmid.The sequence of the gene is confirmed by DNA sequencing.

[0031] To prepare the hG-CSF-L-vF_(γ1) fusion gene, the hG-CSF fragmentis excised from the phGCSF plasmid with HindIII and BamHI and insertedto the 5′-end of the peptide linker in the pL-vFcγ1 plasmid to give thephG-CSF-L-vFcγ1 plasmid. The fusion gene comprising hG-CSF, a 16-aminoacid Gly-Ser peptide linker, and the Fc_(γ1) variant gene is theninserted at the HindIII and EcoRI sites of a mammalian expressionvector, such as pcDNA3 (Invitrogen), as described for thehG-CSF-L-vFc_(γ2) fusion protein. The final expression vector plasmid isdesignated as phGFP1. FIG. 2C shows a fusion gene containing sequencesencoding hG-CSF, a 16-amino acid peptide linker(GlySerGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySer), and the Fc_(γ1)variant with Leu234Val, Leu235Ala and Pro331Ser mutations.

[0032] 4. Expression of the Fusion Protein in Transfected Cell Lines

[0033] Two different rhG-CSF have been produced: a glycosylated formproduced in Chinese Hamster Ovary (CHO) cells and a nonglycosylated formproduced in bacteirial cells. Glycosylated rhG-CSF contains O-linkedoligosaccharides attached to the threonine amino acid residue atposition 133, accounting for approximately 4% of its molecular weight.The carbohydrate chain contributes to the stabilization of the proteinmolecule by suppressing polymerization and conformational changes(Oh-eda et al., J. Biol. Chem., 265:11432-11435, 1990). In in vitrostudies using rhG-CSF, the glycosylated form produced in CHO cells isbiologically more active than the nonglycosylated form produced inbacteirial cells (Nissen, Eur. J. Cancer, 30A Suppl 3:S12-S14, 1994).Furthermore, rhG-CSF derived from CHO cells was shown to beindistinguishable from its natural counterpart in terms of structuralcharacteristics and biological activity (Kubota et al., Biochem.(Tokyo),107:486-492, 1990). In randomized crossover studies in healthyvolunteers, glycosylated rhG-CSF has been found to be 25 to 30% morepotent than the nonglycosylated rhG-CSF on a weight for weight basis inthe mobilization of peripheral blood progenitor cells (see, for example,Hoglund, Med. Oncol., 15:229-233, 1998; Hoglund et al., Eur. J.Haematol., 59:177-183, 1997). To obtain the protein most suitable forclinical use, the hG-CSF-L-vFc fusion protein will be produced in CHOcells as follows.

[0034] The recombinant phGFP1, phGFP2 or phGFP4 expression vectorplasmid is transfected into a mammalian host cell line to achieve theexpression of the hG-CSF-L-vFc fusion protein. For stable high levels ofexpression, a preferred host cell line is CHO cells deficient in theDHUFR enzyme (see, for example, U.S. Pat. No. 4,818,679). A preferredmethod of transfection is electroporation. Other methods, includingcalcium phosphate co-precipitation, lipofectin, and protoplast fusion,can also be used. For electroporation, 10 μg of plasmid DNA linearizedwith BspCI is added to 2 to 5×10⁷ cells in a cuvette using Gene PulserElectroporator (Bio-Rad Laboratories, Hercules, Calif.) set at anelectric field of 250 V and a capacitance of 960 μFd. Two days followingthe transfection, the media are replaced with growth media containing0.8 mg/ml of G418. Transfectants resistant to the selection drug aretested for the secretion of the fusion protein by anti-human IgG FcELISA. Quantitation of the expressed fusion protein can also be carriedout by ELISA using anti-hG-CSF assays. The wells producing high levelsof the Fc fusion protein are subcloned by limiting dilutions on 96-welltissue culture plates.

[0035] To achieve higher levels of the fusion protein expression,co-amplification is preferred by utilizing the gene of DHFR that can beinhibited by the MTX drug. In growth media containing increasingconcentrations of MTX, the transfected fusion protein gene isco-amplified with the DHFR gene. Transfectants capable of growing inmedia with up to 1 μg/ml of MTX are again subcloned by limitingdilutions. The subcloned cell lines are further analyzed by measuringthe secretion rates. Several cell lines yielding secretion rate levelsover about 10, preferably about 30 μg/10⁶ [i.e. million]cells/24h, areadapted to suspension culture using serum-free growth media. Theconditioned media are then used for the purification of the fusionprotein.

[0036] 5. Purification and Characterization of the Fusion Protein

[0037] Conditioned media containing the fusion protein are titrated with1 N NaOH to a pH of 7 to 8 and filtered through a 0.45 micron cellulosenitrate filter. The filtrate is loaded onto a Prosep A columnequilibrated in phospate-buffered saline (PBS). After binding of thefusion protein to Prosep A, the flow-through fractions are discarded.The column is washed with PBS until OD at 280 nm is below 0.01. Thebound fusion protein is then eluted with 0.1 M citrate buffer at pH3.75. After neutralizing with 0.4 volume of 1 M K₂HPO₄, fractionscontaining purified protein are pooled and dialyzed against PBS. Thesolution is then filtered through a 0.22 micron cellulose nitrate filterand stored at 4° C. The molecular weight of purified hG-CSF-L-vFcprotein is in the range of 90 and 110 kDa by SDS-PAGE under non-reducingconditions. Under reducing conditions, the purified protein migratesaround approximately 50 kDa. The fusion protein is quantitated by BCAprotein assay using BSA as the standard.

[0038] 6. In Vitro Biological Assays

[0039] Supernatants of transfectants or purified proteins can be testedfor their ability to stimulate the proliferation of murine myeloblasticNFS-60 cells (Shirafuji et al., Exp. Hematol., 17:116-119, 1989). NFS-60cells are responsive to rhG-CSF but not to rhGM-CSF or hM-CSF. The cellsare maintained in growth medium (RPMI-1640 medium containing 10% FCS andmurine IL-3 at 1 ng/ml). Log phase NFS-60 cells are collected and washedwith assay medium (growth medium without murine IL-3). A total of 1×10⁴cells per sample of NFS-60 in 50 μl is added to each well of a 96-welltissue culture plate. The cells are incubated with 50 μl of assay mediacontaining various concentrations of the hG-CSF-L-vFc fusion protein orthe rhG-CSF control from 0.01 to 100 nM each. The plate is kept at 37°C. and 5% CO₂ in a humidified incubator for 4 days before 10 μl of MTT(2.5 mg/ml in PBS) is added to each well. After 4 h, the cells andformazan are solubilized by adding 100 μl per well of 10% SDS in 0.01 NHCl. The plate is then read at 550 nm with the reference beam set at 690nm. The OD reading is plotted against the concentration of rhG-CSF orthe fusion protein. The inflection point of the sigmoidal curverepresents the concentration at which 50% of the maximal effect, ED50,is induced. The biological activity of hG-CSF-L-vFc relative to that ofrhG-CSF can therefore be compared quantitatively. Preferably, therecombinant fusion proteins should be characterized by and exhibit anenhanced activity of at least 2 fold (2×) relative to that of rhG-CSF ona molar basis. In one embodiment of the present invention, the specificactivity of the hG-CSF-L-vFc fusion protein is in the range of about 1.5to about 6.0×10⁹ units/μmole, compared to about 0.75 to about 3.0×10⁹units/μmole for rhG-CSF based on this cell proliferation assay.

[0040] Supernatants of transfectants or purified proteins can also betested for their ability to stimulate the proliferation anddifferentiation of human bone marrow progenitor cells to form colonies,granulocyte-macrophage colony forming unit (CFU-GM). The procedure is asfollows. Light-density cells from human bone marrow centrifuged overFicoll-Pague are washed and resuspended at 1×10⁶ cells/ml in Iscove'smodified Dulbecco's medium (IMDM) containing 5% FCS. These cellscontaining enriched progenitor cells are incubated in a tissue culturedish overnight at 37° C. and 5% CO₂ to remove all adherent cellsincluding monocytes, macrophages, endothelial cell, and fibroblasts.Cells in suspension are then adjusted to 1×10⁵ cells/ml in IMDMcontaining 5% FCS. For the assay, 0.3 ml of cells, 15 μl of stem cellfactor at 20 μg/ml, 2.4 ml of methylcellulose, and 0.3 ml of mediacontaining several concentrations of hG-CSF-L-vFc (or rhG-CSF control)are mixed. One ml of this cell mixture is plated on a 35-mm petri dish.The dishes are then kept at 37° C. and 5% CO₂ for 10 to 14 d before thecolonies are counted. A dose responsive curve can be plotted against theconcentrations of hG-CSF-L-vFc relative to those of rhG-CSF.

[0041] 7. In Vivo Pharmacokinetic Studies in Rats

[0042] Fisher rats (Harlan Bioproducts for Science, Indianapolis, Ind.)with an average body weight of about 500 g are injected i.v. through thetail vein or s.c. with 100 units of rhG-CSF or the hG-CSF-L-vFc fusionprotein. An equal volume of PBS is injected as a control. Serial 0.5-mlsamples are taken through retro-orbital bleeds at different points (0,0.2, 1, 4, 24, 48, 96, and 168 h) after injection. There are 3 rats foreach time point. Whole blood is collected into tubes containinganticoagulant, cells are removed, and plasma is frozen at −70° C. untilassay is carried out.

[0043] Serum samples are used for NFS-60 cell assays, which measure theactivity of hG-CSF-mediated cell proliferation. A total of 1×10⁴ cellsper sample of NFS-60 in 50 μl is added to each well of a 96-well tissueculture plate. The cells are incubated with 50 μl of assay mediacontaining various concentrations of titrated blood samples. The plateis kept at 37° C. and 5% CO₂ in a humidified incubator for 4 days.Viable cells are stained with 10 μl of MTT (2.5 mg/ml in PBS). After 4h, the cells and formazan are solubilized by adding 100 μl per well of10% SDS in 0.01 N HCl. The plate is then read at 550 nm with thereference beam set at 690 nm. The activities of serum samples areplotted against time points for the calculation of the circulation time.The activity of hG-CSF-L-vFc decreases much slower than that of therhG-CSF control, indicating the longer circulating half-life of thefusion protein in rats.

[0044] The examples described above are for illustration purposes only.They are not intended and should not be interpreted to limit either thescope or the spirit of this invention. It can be appreciated by thoseskilled in the art that many other variations or substitutes can be usedas equivalents for the purposes of this invention, which is definedsolely by the written description and the following claims. TABLE 1Sequences of Oligonucleotides. SEQ ID NO:15′-cccaagcttcccagacccatggctggacct-3′ SEQ ID NO:25′-cggatccgggctgggcaaggtggcgta-3′ SEQ ID NO:3 5′-gagcgcaaatgttgtgtcga-3′SEQ ID NO:4 5′-ggaattctcatttacccggagacaggga-3′ SEQ ID NO:55′-tggttttctcgatggaggctgggaggcct-3′ SEQ ID NO:65′-aggcctcccagcctccatcgagaaaacca-3′ SEQ ID NO:75′-cggatccggtggcggttccggtggaggcggaagcggcggtggaggatcagagcgcaaatgttgtgtcga-3′ SEQ ID NO:8 5′-gagtccaaatatggtccccca-3′ SEQ IDNO:9 5′-ggaattctcatttacccagagacaggga-3′ SEQ ID NO:105′-cctgagttcgcggggggacca-3′ SEQ ID NO:115′-gagtccaaatatggtcccccatgcccaccatgcccagcacctgagtt cgcggggggacca-3′ SEQID NO:12 5′-cggatccggtggcggttccggtggaggcggaagcggcggtggaggatcagagtccaaatatggtccccca-3′ SEQ ID NO:13 5′-gacaaaactcacacatgccca-3′ SEQID NO:14 5′-acctgaagtcgcggggggaccgt-3′ SEQ ID NO:155′-gacaaaactcacacatgcccaccgtgcccagcacctgaagtcgcggg gggaccgt-3′ SEQ IDNO:16 5′-cggatccggtggcggttccggtggaggcggaagcggcggtggaggatcagacaaaactcacacatgccca-3′

1. A recombinant hG-CSF-L-vFc fusion protein comprising hG-CSF, apeptide linker, and a human IgG Fc variant.
 2. The recombinanthG-CSF-L-vFc fusion protein of claim 1, wherein the peptide linkercontains about 20 or fewer amino acids is present between hG-CSF and thehuman IgG Fc variant; and the peptide linker comprises two or more aminoacids selected from the group consisting of glycine, serine, alanine,and threonine.
 3. The recombinant hG-CSF-L-vFc fusion protein of claim1, wherein the human IgG Fc variant comprises a hinge, CH2, and CH3domains of human IgG2 with Pro331Ser mutation.
 4. The recombinanthG-CSF-L-vFc fusion protein of claim 1, wherein the human IgG Fc variantcomprises a hinge, CH2, and CH3 domains of human IgG4 with Ser228Pro andLeu235Ala mutations.
 5. The recombinant hG-CSF-L-vFc fusion protein ofclaim 1, wherein the human IgG Fc variant comprises a hinge, CH2, andCH3 domains of human IgG1 with Leu234Val, Leu235Ala, and Pro331Sermutations.
 6. The recombinant hG-CSF-L-vFc fusion protein of claim 1,wherein the hG-CSF-L-vFc fusion protein is characterized by and exhibitsan enhanced in vitro biological activity of at least 2 fold relative tothat of rhG-CSF on a molar basis.
 7. A CHO-derived cell line producingthe hG-CSF-L-vFc fusion protein of claim 1 in its growth medium inexcess of 10 μg per million cells in a 24 hour period.
 8. TheCHO-derived cell line producing the hG-CSF-L-vFc fusion protein of claim7 in its growth medium in excess of 30 μg per million cells in a 24 hourperiod.
 9. The CHO-derived cell line producing the hG-CSF-L-vFc fusionprotein of claim 1, wherein the human IgG Fc variant comprises a hinge,CH2, and CH3 domains of human IgG selected from the group consisting ofIgG1, IgG2, and IgG4, the IgG Fc contains amino acid mutations toattenuate effector functions, a flexible peptide linker containing about20 or fewer amino acids is present between hG-CSF and human IgG Fcvariant, and the hG-CSF-L-vFc fusion protein is characterized by andexhibits an enhanced in vitro biological activity of at least 2 foldrelative to that of rhG-CSF on a molar basis.
 10. A method for making arecombinant fusion protein comprising hG-CSF, a flexible peptide linker,and a human IgG Fc variant, which method comprises: (a) generating aCHO-derived cell line; (b) growing the cell line under conditions therecombinant fusion protein is expressed in its growth medium in excessof 10 μg per million cells in a 24 hour period; and (c) purifying theexpressed protein from step (b), wherein the recombinant fusion proteinis characterized by and exhibits an enhanced in vitro biologicalactivity of at least 2 fold relative to that of rhG-CSF on a molarbasis.
 11. The method of claim 10, wherein in step (b) growing the cellline under conditions the recombinant fusion protein is expressed in itsgrowth medium in excess of 30 μg per million cells in a 24 hour period.12. The method of claim 10, wherein the flexible peptide linkercontaining about 20 or fewer amino acids is present between hG-CSF andthe human IgG Fc variant; and the flexible peptide linker comprises twoor more amino acids selected from the group consisting of glycine,serine, alanine, and threonine.
 13. The method of claim 12, wherein instep (b) growing the cell line under conditions the recombinant fusionprotein is expressed in its growth medium is in excess of 30 μg permillion cells in a 24 hour period.
 14. The method of claim 10, whereinthe human IgG Fc variant comprises a hinge, CH2, and CH3 domains ofhuman IgG2 with Pro331Ser mutation.
 15. The method of claim 14, whereinin step (b) growing the cell line under conditions the recombinantfusion protein is expressed in its growth medium is in excess of 30 μgper million cells in a 24 hour period.
 16. The method of claim 10,wherein the human IgG Fc variant comprises a hinge, CH2, and CH3 domainsof human IgG4 with Ser228Pro and Leu235Ala mutations.
 17. The method ofclaim 16, wherein in step (b) growing the cell line under conditions therecombinant fusion protein is expressed in its growth medium is inexcess of 30 μg per million cells in a 24 hour period.
 18. The method ofclaim 10, wherein the human IgG Fc variant comprises a hinge, CH2, andCH3 domains of human IgG1 with Leu234Val, Leu235Ala, and Pro331Sermutations.
 19. The method of claim 18, wherein in step (b) growing thecell line under conditions the recombinant fusion protein is expressedin its growth medium is in excess of 30 μg per million cells in a 24hour period.
 20. A method for making a recombinant fusion proteincomprising hG-CSF, a flexible peptide linker, and a human IgG Fcvariant, which method comprises: (a) generating a CHO-derived cell line;(b) growing the cell line under conditions the recombinant protein isexpressed in its growth medium in excess of 10 μg per million cells in a24 hour period; and (c) purifying the expressed protein from step (b),wherein the recombinant fusion protein is characterized by and exhibitsan enhanced in vitro biological activity of at least 2 fold relative tothat of rhG-CSF on a molar basis; wherein the flexible peptide linkercontaining about 20 or fewer amino acids is present between hG-CSF andthe human IgG Fc variant; and the flexible peptide linker comprises twoor more amino acids selected from the group consisting of glycine,serine, alanine, and threonine; and wherein the human IgG Fc variantcomprises a hinge, CH2, and CH3 domains selected from the groupconsisting of human IgG2 with Pro331Ser mutation, human IgG4 withSer228Pro and Leu235Ala mutations, and human IgG1 with Leu234Val,Leu235Ala, and Pro331Ser mutations.